SIRT1 Attenuates LPS-induced Barrier Dysfunction Via Inhibiting P53 And Protecting ?-catenin

Sepsis is a life-threatening inflammatory response syndrome caused by imbalance of host response to infection.Its pathophysiological process is complex,clinical symptoms are diverse,and it consumes a lot of medical resources.Once the patient has sepsis,the prognosis is poor.Unfortunately,our mechanism for sepsis is not yet fully understood and lacks effective treatment.In recent years,many studies have shown that the destruction of blood vessel barrier function in the occurrence and development of sepsis play a role in promoting.The integrity of vascular endothelial cells is one of the key factors to maintain vascular barrier function.Dysfunction of endothelial cells leads to the destruction of barrier function,which leads to increased vascular permeability and leakage of intravascular substances into tissue spaces,resulting in tissue edema and organ dysfunction.When sepsis causes acute lung injury,vascular endothelial cells are damaged,pulmonary microvascular leakage increases and the ratio of dry to wet increases.Stable endothelial cell junction can alleviate lung injury after sepsis and reduce mortality in sepsis patients.Studies have shown that lipopolysaccharide(LPS)derived from the cell wall of Gram-negative bacteria can damage endothelial cells,leading to vascular endothelial barrier dysfunction,so that a variety of inflammatory factors,complements,proteins and other macromolecules in the blood permeate through the damaged endothelium outside the blood vessels,resulting in the body of patients with Effective circulating blood volume decreased significantly,but the mechanism of LPS-induced impairment of microvascular endothelial cell barrier function in sepsis remains unclear.Therefore,the mechanism of endothelial barrier dysfunction may provide a potential breakthrough for the treatment of sepsis.The barrier function of endothelial cells depends on many factors,including cell transmembrane pathway and cell bypass pathway.Cell bypass pathways depend on the balance between cell-to-cell junctions and the contractile forces of cytoskeleton proteins,which include the forms of adhesion and tight junctions.Endothelial-specific adhesion junction-the assembly and construction of vascular endothelial cadherin precedes tight junction formation,and plays an important role in maintaining vascular integrity and endothelial function.Catenin is a group of adhesion proteins with Cadherin acting on the cell membrane,including a,beta,gamma and so on.Beta-catenin is expressed in endothelial cells,and plays an important role in the regulation of cell proliferation,differentiation and apoptosis.Beta-catenin,located on the cell membrane,plays a regulatory role in the interaction between cytoskeleton protein and E-cadherin,adhesion between homologous cells and motility,maintaining cell adhesion and preventing cell movement.When the adhesion junction structure is damaged,the contraction force of the skeleton protein from the centripetal force of F-actin forming stress fibers will be greater than the adhesion force between cells,the balance between the two will be destroyed,the gap between cells will increase,and the permeability will increase.Therefore,decreased expression of beta-catenin protein will destroy the bypass pathway of endothelial cells and lead to the destruction of endothelial cell barrier.Many inflammatory factors,including LPS,can damage the endothelial cell barrier,suggesting that decreased expression of beta-catenin protein promotes the increase of vascular permeability after sepsis.Silence information regulator 2 homolog 1(SIRT1)is a very important histone/non-histone deacetylase recently discovered.So far,SIRT1 has been widely studied.SIRT1 mainly increases or inhibits the activity of the target protein by deacetylation,and plays an important role in regulating cell differentiation,cell metabolism,cell cycle,apoptosis and stress response.Increasing evidence suggests that increased expression or activity of SIRT1 proteins may prolong the survival of septic animal models,and its role may be related to improving vascular permeability.Studies have found that downregulation of SIRT1 protein aggravates vascular permeability after sepsis.Interestingly,SIRT1 agonists reversed LPS-induced increased vascular permeability,suggesting that increased SIRT1 protein expression or activation may reduce vascular permeability after sepsis.However,the role of SIRT1 in endothelial cells,which are important for vascular permeability,has not been fully understood and the mechanism remains to be elucidated.As we all know,p53 is the first non histone protein modified by SIRT1 deacetylation.P53 protein plays an important role in promoting apoptosis and inhibiting tumor growth.P53 protein is affected by post-translational modifications such as phosphorylation and acetylation,in which p53 acetylation leads to an increase in p53 activity.Our previous study showed that SIRT1 protein expression decreased and activity decreased after severe hemorrhagic shock,resulting in increased deacetylation of p53 and increased apoptosis of renal tubular epithelial cells;activation of SIRT1 can reduce apoptosis of renal tubular epithelial cells after shock by up-regulating SIRTl-deacetylated p53 signaling pathway.Other researchers have shown that SIRT1 deacetylated p53 can block oxidative stress such as hypoxia-reoxygenation,ischemia,hyperglycemia and shear stress induced endothelial cell aging and barrier dysfunction.Studies have shown that p53 can reduce the expression of beta catenin(beta-catenin).It is noteworthy that the basal level of p53 in non-stress cells does not cause the down-regulation of beta-catenin protein,but the high level of activated p53 has a strong inhibitory effect on the activity of beta-catenin protein under extensive genotoxic stress.SIRT1 deacetylated p53 significantly reduced p53 activity.Therefore,we speculate that inactivation of deacetylase SIRT1 leads to an increase in p53 acetylation,promotes adhesion injury involving beta-catenin,increases endothelial cell permeability,and thus aggravates vascular endothelial barrier dysfunction in LSP-stimulated septic cells;and an increase in SIRT1 expression or activity can deacetylate p53.Thus reversing the process,protecting beta-catenin and alleviated LPS induced dysfunction of vascular endothelial barrier.ObjectIn this study,C57 mice and human umbilical vein endothelial cells(HUVECs)were used to investigate whether SIRT1 inhibits the down-regulation of the expression of beta-catenin by deacetylating p53,thus protecting the adhesion junction and protecting the function of vascular endothelial barrier,which will help to elucidate further.To explore the mechanism of sepsis-mediated increased permeability of microvascular endothelial cells and provide new theoretical basis for the prevention and treatment of endothelial cell barrier dysfunction in sepsis.MethodsThis study was carried out by animal model and cell model,one of which was the C57 mouse model,and the other was the human umbilical vein endothelial cell model.Primary identification and isolation,cell culture,Western blot,immunofluorescence,transendothelial electrical resistance(TER)and fluorescein isothiocyanate labelled dextran(FITC-dextran)leakage coefficients were measured.The expression of SIRT1 and its effect on the expression of downstream beta-catenin and p53 acetylation were studied by means of mesenteric microvascular leakage.SIRT1 specific agonist SRT1720 or SIRT1 specific inhibitor EX527 were used to increase or inhibit the activity of SIRT1,SIRT1 siRNA was used to down-regulate the expression of SIRT1 protein,and the levels of SIRT1 protein,beta-catenin and acetylated p53 protein were detected by Western blotting.The level of fluorescence and intracellular distribution of beta-catenin.The resistance between endothelial cells was measured by electrical impedance meter,and the leakage of endothelial cells was detected by FITC-dextran leakage method,which indirectly reflected the barrier function of endothelial cells.In C57 mice,FITC-dextran was injected into the jugular vein,and then the leakage of FITC-dextran from the mesenteric venules was observed.In this study,the average(standard error)was used to analyze the data.The statistical package for the social science(SPSS)19.0 software was used to analyze the data.One-way analysis of variance(ANOVA)was used to analyze the differences between groups(LSD method was used for homogeneity of variance,and variance was not homogeneous sometimes).Using Dunnect's T3 method),P<0.05 showed statistical difference.Research groups and Results1.Activation of SIRT1 reduces LPS-sparked increased permeability in mesentery venulesObj ective to study the effect of SIRT1 on the mesenteric microvascular endothelial cells induced by LPS in mice with sepsis.They were divided into Control group,LPS+solvent group(LPS+Vehicle)group and LPS+SRT1720 group.The control group was injected with normal saline,the LPS+Vehicle group was injected with the same amount of solvent as SRT1720,and LPS was injected into the abdominal cavity 2 hours later;the LPS+SRT1720 group was injected with SIRT1 agonist SRT1720(10mg/kg)into the tail vein,and LPS was injected into the abdominal cavity 2 hours later.Six hours later,the jugular veins of mice were cannulated,mesenteric venules were observed under microscope,and FITC-dextran was injected into the jugular veins.The leakage of FITC-dextran from the mesenteric venules was observed.The result showe that:LPS stimulated vascular permeability increased,showing a large number of fluorescent exudation(P<0.05),while SIRT1 agonist SRT1720 pretreatment could significantly improve LPS induced mesenteric microvascular leakage increased.2.LPS induces down-regulation of SIRTl protein in time-dependent manner.The cells were stimulated with LPS of 500 ng/mL.The time points of SIRT1 protein determination were 1,2,4,8,12 and 24 hours after LPS stimulation.Results:The SIRT1 protein level was significantly down-regulated after 1 h(P<0.05),and the low level of SIRT1 protein was maintained until 24 h.3.SIRT1 inhibits LPS-induced down-regulation of ?-cateninThe sepsis cell model was constructed and pretreated with SIRT1 agonist SRT1720 and inhibitor EX527 respectively to investigate the effect of SIRT1 on beta catenin protein.The agonists were divided into Control group,SRT1720 group,LPS+solvent group(LPS+Vehicle)group and LPS+SRT1720 group.The inhibitor group was divided into Control group,EX527 group,LPS+solvent group(LPS+Vehicle)group,LPS+EX527 group.Control group only changed the same amount of medium as the other groups;SRT1720 group added 5 micromol/L SIRT 1 agonist SRT1720,treated endothelial cells 24 hours later,changed the medium;EX527 group added 20 micromol/L SIRT 1 inhibitor EX527,treated endothelial cells 24 hours later,changed the medium;The endothelial cells were stimulated by 500 ng/ml LPS for one hour after 24 hours,and the endothelial cells were treated with 5 micromol/L SIRT1 agonist SRT1720 for 24 hours,followed by 500 ng/ml LPS stimulation for one hour.In LPS+EX527 group,the endothelial cells were pretreated with 20 micromol/L SIRT inhibitor EX527 for 24 hours,and then stimulated with 500 ng/ml LPS for 1 hour.The expression of beta-catenin protein was detected by immunofluorescence and immunofluorescence.Results:SIRT1 agonist SRT1720 had no significant effect on the level of beta-catenin protein in normal cells;SIRT1 inhibitor EX527 could induce the decrease of beta-catenin protein in normal cells(P<0.05);the level of beta-catenin protein decreased after LPS stimulation(P<0.05);and the pretreatment with SIRT1 agonist SRT1720 could significantly improve LPS-induced cell proliferation.The level of beta-catenin protein was down regulated(P<0.05).Pretreatment with SIRT1 inhibitor EX527 could further aggravate the decrease in the level of beta-catenin protein(P<0.05).4.Activation of SIRT1 inhibits the increase in LPS-induced p53 acetylation activityThe grouping is the same as 3.Acetylation level of p53 was measured.RESULTS:SIRT1 agonist SRT1720 had no effect on the acetylation level of p53 in normal cells,LPS stimulated cells increased the acetylation level of p53(P<0.05),and pretreatment with SIRT1 agonist SRT1720 decreased the acetylation level of p53.5.Inhibition of p53 reduced the permeability of endothelial cells induced by LPSThey were divided into control group,PFT-alpha group,LPS+Vehicle group and LPS+PFT-alpha group,which were basically the same as 3.1 except that the pretreatment drugs were replaced by p53 inhibitor PFT-alpha.The concentration of PFT-alpha was 30 mol/L.Cell TER levels and FITC-dextran leakage were detected.Results:p53 inhibitor PFT-alpha did not affect the TER level and FITC-dextran leakage of normal cells;LPS-stimulated cells resulted in the decrease of TER level and the increase of FITC-dextran leakage(P<0.05);p53 inhibitor PFT-alpha pretreatment could reduce the decrease of TER level and the increase of FITC-dextran leakage caused by LPS(P<0.05).6.p53 participates in the down-regulation of ?-catenin induced by LPS.The grouping is the same as 5.The expression of beta-catenin protein was measured by immuno imprinting.Results:PFT-alpha,a P53 inhibitor,had no significant effect on the level of beta-catenin protein in normal cells,while pretreatment with PFT-alpha,a p53 inhibitor,inhibited the down-regulation of beta-catenin s induced by LPS(P<0.05).ConclusionActivation of the deacetylase SIRT1 attenuates the increased permeability of endothelial cells after LPS stimulation,and the mechanism is related to SIRT1 deacetylating p53 to inhibit its activity,thereby reducing the down-regulation of ?-catenin.